Method for carrying out the ex vivo expansion and ex vivo differentiation of multipotent stem cells

ABSTRACT

The invention relates to a method for carrying out the expansion of multipotent stem cells ex vivo. Moreover, the invention relates to a two-stage method for carrying out the expansion and differentiation of multipotent stem cells ex vivo, in which it is possible for the stem cells to be gene transfected during the first stage, i.e. during the expansion phase. In this phase, the differentiation of the multipotent stem cells takes place optionally in cells of the hematopoietic, endothelial or mesenchymal cell lineage. Stem and progenitor cells as well as mature cells of the hematopoietic, endothelial and mesenchymal cell lineage, which are obtained in this way, can be used, among other things, for the prophylaxis, diagnosis and therapy of human deseases and for tissue engineering.

The invention relates to a method for carrying out the expansion ofmultipotent stem cells ex vivo. Moreover, the invention relates to atwo-stage method for carrying out the expansion and differentiation ofmultipotent stem cells ex vivo, in which it is possible for the stemcells to be gene transfected during the first stage, i.e. during theexpansion phase. In the second phase, the differentiation of themultipotent stem cells takes place optionally in cells of thehematopoietic, endothelial or mesenchymal cell lineage. Stem andprogenitor cells as well as mature cells of the hematopoietic,endothelial and mesenchymal cell lineage, which are obtained in this waycan be used, among other things, for the prophylaxis, diagnosis andtherapy of human diseases and for tissue engineering.

BACKGROUND AND STATE OF THE ART

a) Endothelial Cell Lineage

The establishment and maintenance of a vessel supply are an absoluteprecondition for the growth of normal and malignant tissue. Twoprocesses are basically responsible for neovascularisation. The firstprocess, namely vasculogenesis, involves the in situ differentiation ofhemangioblastoma to form endothelial cells and their subsequentorganisation into a primary capillary plexus. (Risau et al., Development102, 471-478, 1988; Risau et al., Annu. Rev. Cell Dev. Biol. 11, 73-91,1995). The second process, the so-called angiogenesis, is defined as theformation of new blood vessels by budding of existing blood vessels.Previously, it had been assumed that the process of vasculogenesis takesplace only during the early embryonal phase whereas angiogenesis takesplace both prenatally and postnatally. The hemangioblast as a joint stemcell for hematopoietic cells and endothelial cells has recently beenidentified as a transient cell stage which is detectable for a briefperiod only during the early embryo development. Subsequently, thehemangioblastoma seems to differentiate out without renewing itself(Choi et al., Development 125, 725-732, 1998). However, it must bepointed out that these results are based on animal experiment studiesand cannot necessarily be transferred to the human system. It would beconceivable that, in man, hemangioblastoma or other subpopulations ofendothelial progenitor cells persist beyond the embryonal phase andcould, in the adult organism, circulate, differentiate and participatein the formation of new blood vessels. This hypothesis has meanwhilebeen supported by numerous studies (Asahara et al., Science 275,964-967, 1997; Yin et al., Blood 90, 5002-5012, 1997; Shi et al., Blood92, 362-367, 1998; Takahashi et al., Nature Med. 5, 434-438, 1999;Asahara et al., Circul. Res. 85, 221-228, 1999; Lin et al., J. Clin.Invest. 105, 71-77, 2000; Kalka et al., Circul. Res. 86, 1198-1202,2000; Kalka et al., Ann. Thorac. Surg. 70, 829-834, 2000; Bhattacharyaet al., Blood 95, 581-585, 2000; Crosby et al., Circul. Res. 87,728-730, 2000; Murohara et al., J. Clin. Invest. 105, 1527-1536, 2000;Peichev et al., Blood 95, 952-958, 2000).

It has been possible to show that the population of adult humanAC133-positive stem and progenitor cells contains endothelial progenitorcells (EPC) which, in the presence of SCGF, a hematopoietic stem cellgrowth factor, and VEGF, an angiogenic cytokine, differentiate to formfunctional intact endothelial cells which are capable of forming bloodvessels in vivo (Gehling et al., Blood 95, 3106-3112, 2000).

In vitro, the rate of proliferation of endothelial cells is normallyvery low. Endothelial progenitor cells, too, exhibit only a low growthtendency in conventional cell culture media. The cell counts such asthey would be required for many clinical applications could not beachieved in this way. Culture conditions allowing an ex vivo expansionof endothelial progenitor cells and endothelial cells have so far notbeen developed. Not even with the culture conditions selected in theabove-mentioned study (Gehling et al., loc. cit.) has it been possibleto induce proliferation of the endothelial progenitor cells in the senseof an expansion. It has merely been possible to achieve a maximum 8-foldmultiplication of these progenitor cells. In order to obtain the cellcounts of endothelial progenitor cells necessary for clinicalapplications, however, a hundred-fold expansion needs to be aimed at.

Quirici et al. (Br. J. Heamatol. 115, 186-194, 2001) has described aprocess for the expansion of endothelial cells in which processCD133-positive bone marrow cells were cultivated initially for 3-4 weeksin the presence of VEGF, bFGF and IGF-1. After purification using Ulexeuropaeus agglutinin-1 (UEA-1), a further cultivation phase (3-4 weeks)with VEGF, bFGF and IGF-1 follows. Although an approximately 2300 foldexpansion is described, it should be noted that the cells purified byUEA-1 represent only approximately 0.5% of the original cells as aresult of which the process is practicable only in extremely limitedcases, particularly also because of the cultivation phases of a total of6-8 weeks which is thus very long.

Against this background, the need for an expansion process persistswhich—preferably starting out from multipotent stem cells obtained bytaking blood—provides a sufficient expansion rate and can be carried outwithout major technical or time expenditure. By providing such aprocess, numerous new applications would be opened up which have notbeen feasible previously because the cell material required for thispurpose could not be provided at all or in insufficient quantities.

Thus the possible fields of application of endothelial progenitor cells(EPC) and endothelial cells (EC) expanded ex vivo are numerous. In thisrespect, diagnosis, prophylaxis and therapy of cardiovascular andmalignant (such as e.g. neoplastic) diseases and tissue engineering canbe mentioned, on the one hand. An example in the field of cardiology isthe direct introduction of EPC in underperfused vascular areas of theheart in order to induce the formation of new blood vessels. This methodcould be transferred to perfusion problems in other organs and areas ofthe body. In the area of tissue engineering, EPC can be used to producenew blood vessels in vitro for clinical purposes. Moreover, the EPC canserve the purpose of facilitating the supply of vessels in the case ofskin transplants and artificially produced (tissue engineered) organssuch as liver and pancreas. A further field of application is the use ofgene transfected EPC as a vehicle for certain gene products. Thesegenetically modified EPCs can be introduced into the vessels of diseasedorgans and tumours both for diagnostic and for therapeutic purposes.

Consequently, it is a task of the present invention to provide anexpansion method which—preferably starting out from multipotent stemcells obtained by taking blood—provides a sufficient expansion rate andcan be carried out without major technical or time expenditure.

b) Hematopoietic Cell Lineage

Since the beginning of the nineties, patients suffering from a malignanttumour have been treated on an increasing scale with high dosagechemotherapy. Since the main side effect consists of an enduring bonemarrow aplasia, this therapy is carried out in combination with anautologous transplant of hematopoietic progenitor cells. To obtain asufficient number of hematopoietic progenitor cells, is necessary toeither carry out a large volume removal of bone marrow under fullanaesthesy or to carry out one to three leukaphereses.

It is possible for the stem cell reserve of a patient to be insufficientto obtain the quantity of progenitor cells necessary for transplantionfrom the bone marrow or the peripheral blood. In the last ten years,numerous working groups have worked on the development of cultureconditions allowing an ex vivo expansion of hematopoietic progenitorcells (Berenson et al., Blood 77, 1717-1722, 1991; Brandt et al., Blood79, 634-641, 1992; Haylock et al., Blood 80, 1405-1412, 1992; Brugger etal., Blood 81, 2579-2584, 1993; Sato et al., Blood 82, 3600-3609, 1993;Rice et al., Exp. Hematol. 23, 303-308, 1995; Alcorn et al., J. Clin.Oncol. 14, 1839-1847, 1996; Peters et al., Blood 87, 30-37; 1996,Yonemura et al., Blood 89, 1915-1921, 1997; Reiffers et al., Lancet 354,1092-1093, 1999, McNiece et al., Blood 96, 3001-3007; 2000). By means ofan expansion culture of the progenitor cells preceding thetransplantation, it would be possible to reduce the quantity of bonemarrow aspirate or leukapheresate to a minimum. For patients with alimited stem cell reserve, a satisfactory progenitor cell transplantcould be produced. Moreover, it is hoped that the transplantation ofprogenitor cells expanded ex vivo would provide a more rapidreconstitution of hematopoeisis.

So far, however, none of the working groups mentioned above has beenable to present culture conditions which cause an ex vivo expansion ofthe “true” hematopoietic stem cell. Instead, the culture conditionsdeveloped so far lead to an early differentiation of the stem cells andconsequently to an ex vivo expansion of determined progenitor cells,which have lost their stem cell characteristics. These cells are notsuitable for transplanting since they are not able to regenerate apermanent hematopoiesis following myeloablative chemotherapy (Shih etal., J. Hematother. Stem Cell Res. 9, 621-628, 2000, McNiece et al.,Exp. Hematol. 29, 3-11, 2001).

A further task of the present invention consequently consists ofdeveloping a process whose culture conditions permit an ex vivoexpansion of transplantable hematopoietic stem cells.

c) Mesenchymal Cell Lineage

In the area of the mesenchymal cell lineage, Reyes et al. (Blood 96,2615-2625, 2001) has described an ex vivo expansion method in whichCD45/glycophorin-A-negative mononuclear bone marrow cells are cultivatedin the presence of EGF and PDGF-BB. However, a disadvantage of thisprocess—as in the process described already by Quirici et al. (compareabove) for the endothelial cell lineage—consists in that the mononuclearbone marrow cells used represent only a portion of 0.1 to 0.5% of thebone marrow cells. Consequently, this entails additional purificationand enrichment stages. Moreover, it must be considered that theCD45⁻/GlyA⁻ cells exhibit only a very low proliferation rate. Thus, thecell reduplication rate is 46-60 hours. Starting out from a removal of100 ml of bone marrow, 1×10⁸ mononuclear blood cells are obtained ofwhich 0.1-0.5% are CD45⁻/GlyA⁻, i.e. 1-5×10⁵ CD45⁻/GlyA⁻cells. After 14days in the culture, only 6.4×10⁶ to 3.2×10⁷ stem cells are thusobtained. For this reason, the expansion method according to Reyes etal. is of only little practical importance, in particular for clinicaluse.

The Present Invention:

The task of the present invention consists of avoiding the disadvantagesknown from the state of the art and of providing an expansion method bymeans of which markedly higher cell counts can be achieved duringexpansion than has been possible so far in the state of the art. Inparticular, a method is to be provided by means of which it is possibleto multiply, in a controlled manner, progenitor cells and mature cellsof different cell lineages (hematopoietic, endothelial and mesenchymalcell lineages) equally in different differentiation stages. The methodshould be practicable without any major technical or time expenditureand be preferably based on multipotent stem cells accessible by thesimple taking of blood.

According to the invention, the task is achieved by way of methods forcarrying out the expansion of multipotent stem cells in whichmultipotent stem cells are cultivated in the presence of Flt3 ligand andat least one growth factor from the group consisting of SCF, SCGF, VEGF,bFGF, insulin, NGF and TGF-β. In each case, IGF-1 and/or EGF canoptionally additionally be used.

According to a particular embodiment, one of the following combinationsis chosen:

-   -   a) Flt3 ligand and VEGF,    -   b) Flt3 ligand, SCGF and VEGF,    -   c) Flt3 ligand and EGF,    -   d) Flt3 ligand, EGF and bFGF,    -   e) the growth factors mentioned in a) to d) in combination with        IGF-1 and/or EGF.

Surprisingly enough, it is possible by using the above-mentioned growthfactors to achieve a more than hundred fold multiplication of the cellcounts used to obtain, starting out, for example, from only 50 ml ofleukapheresis product 1×10⁹ to 1×10¹⁰ multipotent stem cells alreadyafter a 14 day culture. The expansion can thus be carried out on amarkedly larger scale than in the state of the art. A further advantage,consists of the fact that it is possible to use sources for stem cellswhich, such as e.g. blood, are obtainable in a simple manner.Unexpectedly, the use of Flt3 ligand, which is a hematopoietic growthfactor, in combination with the above-mentioned growth factors do notlead to a premature differentiation of the stem cells, not even in thedirection of the hematopoietic cell lineage. This has the particularadvantage that it is possible to allow the multipotent stem cells tomature after expansion in a subsequent differentiation phase. At thesame time, the separation of expansion and differentiation according tothe invention makes it possible, in an advantageous manner, to effect agenetically engineered modification of the still multipotent stem cells.In other words, it is possible to transfect the stem cells, while theyare strongly proliferating, with vectors which preferably containnucleic acid sequences encoding proteins or polypeptides which are notnaturally expressed in these cells.

Since it has come to light that the use of Flt3 ligand promotesdifferentiation in the presence of VEGF and bFGF, this combinationshould be avoided if an expansion of multipotent stem cells is to beaimed at exclusively, i.e. without or without significantdifferentiation. Instead, it is possible according to the invention forthe differentiation of the expanded multipotent stem cells to take placein the subsequent second step by means of which a differentiation can becarried out in a targeted manner into one of the three cell lineages(endothelial, hematopoietic and mesenchymal).

Consequently, the subject matter of the invention also consists of atwo-phase method (two-phase culture system) in which multipotent stemcells are expanded and developed to produce human progenitor cells andmature cells of the hematopoietic, endothelial and mesenchymal celllineage. The above-mentioned expansion process according to theinvention corresponds to phase I of the two-phase method. Consequently,for simplification, reference will be made in the following to phase I,the details given also applying equally to the expansion method (i.e.without subsequent differentiation phase).

Consequently, the invention also relates to a method for carrying out invitro (ex vivo) expansion and differentiation of multipotent stem cellsin which

-   -   a) the expansion process according to the invention is carried        out in a first phase for the expansion of multipotent stem cells        (i.e. in the presence of Flt3 ligand and at least one growth        factor from the group consisting of SCF, SCGF, VEGF, bFGF,        insulin, NGF and TGF-β (if necessary in combination with IGF-1        and/or EGF in each case) and    -   b) the expanded cells are differentiated in a second phase, the        cells being        -   (i) cultivated for (the induction of) hematopoietic            differentiation in the presence of at least one growth            factor from the group consisting of G-CSF, GM-CSF, M-CSF,            IL-3, IL-6, IL-11, TPO and EPO, optionally in combination            with at least one growth factor from the group consisting of            IL-1, SCF and SCGF,        -   (ii) cultivated for (the induction of) endothelial            differentiation in the presence of at least one growth            factor from the group consisting of VEGF, aFGF, bFGF, ECGS,            AP-1, AP-2, NGF, CEACAM, pleiotrophin, angiogenin, PlGF, and            HGF, optionally in combination with at least one growth            factor from the group consisting of LIF, EGF, IGF-1, PDGF,            PDECGF, TGFα, TGFβ, TNFα, estrogen, proliferin, IL-3, G-CSF,            GM-CSF, EPO SCF and SCGF,        -   (iii) cultivated for (the induction of) mesenchymal            differentiation in the presence of at least one growth            factor from the group consisting of PDGF-BB, TGF-β and            BMP-4, optionally in combination with at least one growth            factor from the group consisting of EGF, aFGF, bFGF, IGF-1,            SCF and SCGF,        -   (iv) cultivated for (the induction of) neuronal            differentiation in the presence of at least one growth            factor from the group consisting of NGF, CNTF, GDNF and            BDNF, optionally in combination with at least one growth            factor from the group consisting of EGF, bFGF, IGF-1, IL-1b,            Il-6, Il-11, LIF, Flt3 ligand, SCF and BMP-4, or        -   (v) cultivated for (the induction of) hepatocytic            differentiation in the presence of HGF, optionally in            combination with at least one growth factor from the group            consisting of EGF, IGF-1, insulin, HCG, KGF, TNF-α, Flt3            ligand, SCF and SCGF.

To carry out the expansion (in line with the first phase of thetwo-stage method), the use of Flt3 ligand in the following combinationis preferred:

-   -   a) Flt3 ligand and VEGF,    -   b) Flt3 ligand, SCGF and VEGF,    -   c) Flt3 ligand and EGF,    -   d) Flt3 ligand, EGF and bFGF,    -   e) the growth factors mentioned in a) to d) in combination with        IGF-1 and/or EGF.

In the second phase, use is made according to a particular embodiment

-   -   (i) of a combination of SCF, IL-3, IL-6, G-CSF, GM-CSF and EPO        for the hematopoietic differentiation    -   (ii) of a combination of SCGF and VEGF for the endothelial        differentiation    -   (iii) of a combination of EGF, PDGF-BB, IGF-1, bFGF and BMP-4        for the mesenchymal differentiation    -   (iv) of a combination of BDNF, GDNF, EGF and bFGF for the        neuronal differentiation or    -   (v) of a combination of Flt3 ligand, SCF, HGF and TGF-β for the        hepatocytic differentiation.

In a particularly easy manner, the method can additionally be used for agene transfection of the stem cells without impeding cell expansion. Thegene transfected stem cells can be differentiated into thehematopoietic, endothelial and mesenchymal cell lineage in a manneranalogous to the genetically non-modified stem cells.

During gene transfection, a nucleic acid sequence that codes for aprotein or polypeptide not naturally expressed in the cells (in thefollowing referred to as “foreign gene”) is introduced.

The multipotent stem cells can be obtained from mobilised ornon-mobilised autologous peripheral blood or bone marrow of the patientor from the blood from the veins of the umbilical cord. The mobilisationtherapy can consist of a subcutaneous or intravenous injection of growthfactors such as G-CSF, GM-CSF or SCF and/or intravenous or oralapplication of cytostatics. Obtaining the multipotent stem cells fromG-CSF mobilised peripheral blood (freshly obtained or frozenleukapheresis products) represents a particular embodiment of theinvention. The multipotent stem cells can be obtained in the mononuclearcell fraction. By using antibodies which recognise special antigens tomultipotent stem cells, it is possible to isolate the multipotent stemcells. The following antibodies can be used: anti-CD7 mAb, anti-CD31 mAb(PECAM-1), anti-CD34 mAb, anti-CD54 (ICAM-1) mAb, anti-CD90 (Thy-1) mAb,anti-CD114 (G-CSF-R) mAb, anti-CD116 (GM-CSF-R) mAb, anti-CD117 (c-kit)mAb, anti-CDw123 (IL-3R α chain) mAb, anti-CD127 (IL-7R) mAb, anti-AC133mAb, anti-CD135 (Flk3/Flk2) mAb, anti-CD140b (PDGF-Rβ) mAb, anti-CD144(VE-cadherin) mAb, anti-CD164 mAb, anti-CD172a mAb, anti-CD173 mAb,anti-CD174 mAb, anti-CD175 mAb, anti-CD176 mAb, anti-CD184 (CXCR4) mAb,anti-CD201 (endothelial cell protein C receptor) mAb, anti-CD202b(Tie-2/Tek) mAb, anti-CD224 mAb, anti-CD227 (MUC-1) mAb, anti-CD228 mAb,anti-CD243 (MDR-1) mAb, anti-EGF-R mAb, anti-FGF-R mAb, anti-P1H12 mAb,anti-KDR mAb, anti-EN4 mAb, anti-BENE mAb. As an alternative toantibodies, lectins such as e.g. Ulex europaeus agglutinin-1 can be usedfor the selection of the multipotent stem cells. In addition, themultipotent stem cells can also be obtained by depletion. For thispurpose, mAb CD45 can be used. In principle, the multipotent stem cellscan be obtained in the following cell populations: AC133⁺ CD34⁺, AC133⁺CD34⁻, AC133⁻ CD34⁻. The selection of the overall population ofAC133-positive stem cells and progenitor cells is recommended.

The individual phases of the culture system according to the inventionwill be explained in further detail in the following:

Phase I of the Culture System: Expansion Phase

After obtaining the multipotent stem cells, these cells are expanded exvivo in suspension cultures. IMDM, MEM, DMEM, X-Vivo10, RPMI, M-199medium, EGM-2 can be used as basal medium. The basal medium can besupplemented with fetal calf serum, horse serum or human serum. As analternative, the multipotent stem cells can be expanded serum-free. Forthe expansion phase, the above-mentioned (preferably recombinant) humangrowth factors an be used. Moreover, the medium can be supplemented withhydrocortisone. The ex vivo expansion of the multipotent stem cells inIMDM+10% FBS+10% horse serum+10⁻⁶ mol/l hydrocortisone+SCGF+Flt3ligand+VEGF represents a preferred embodiment according to theinvention. SCGF can be replaced by SCF without problem.

During the expansion phase, genetic material can be transferred to themultipotent stem cells. The genetic material which is transferred to themultipotent stem cells expanded ex vivo can be genes which encodenumerous proteins. These genes comprise those encoding fluorescentproteins such as e.g. GFP. Moreover, these genes also comprise thoseencoding different hormones, growth factors, enzymes, cytokines,receptors and antitumour substances. In addition, the genes can encode aproduct which controls the expression of another gene product or geneswhich block one or several steps of a biological reaction sequence. Inaddition, the genes can encode a toxin which is fused with apolypeptide, e.g. a receptor ligand, or with an antibody which binds thetoxin to the target cell. Correspondingly, the gene can encode atherapeutic protein which is fused with a “targeting” polypeptide inorder to transfer a therapeutic effect onto a diseased organ or tissue.

The nucleic acids are introduced into the multipotent stem cellsexpanded ex vivo by means of a method which guarantees theirincorporation and expression in the stem cells. These methods cancomprise vectors, liposomes, naked DNA, electroporation etc.

Phase II of the Culture System: Differentiation Phase

In suspension cultures, the multipotent stem cells can be differentiateddirectly after isolation or after prior ex vivo expansion, geneticallynatively or in a modified manner into the hematopoietic, endothelial ormesenchymal cell lineage. The following media can be used as basalmedium: IMDM, MEM, RPMI, M-199, X-Vivo10, EGM-2, Williams medium E, SATOmedium, DMEM or DMEM-F12. The basal medium can be supplemented withfetal calf serum, horse serum or human serum. As an alternative,serum-free culture conditions can be used.

In order to induce a hematopoietic differentiation, the following(preferably recombinant) human growth factors are added: G-CSF, GM-CSF,M-CSF, IL-3, IL-6, IL-11, TPO and/or EPO. Additionally, one or severalof the following (preferably recombinant) human growth factors can beused: IL-1, SCF and SCGF. The use of SCF, IL-3, IL-6, G-CSF and TPO incombination with EPO represents a particularly preferred embodiment ofthe invention.

The induction of the differentiation of the multipotent stem cells intothe endothelial cell lineage, i.e. into endothelial progenitor cells andinto mature endothelial cells is achieved by using the following(preferably recombinant) human growth factors: VEGF, bFGF and/or ECGS.Additionally, one or several of the following (preferably recombinant)human growth factors can be used: AP-1, AP-2, LIF, EGF, IGF-1, NGF,CEACAM, HGF, SCF and SCGF. The use of SCF, VEGF, bFGF, IGF-1, EGF, LIFplus AP-1 represents an embodiment preferred according to the invention.

In order to induce mesenchymal differentiation, the following(preferably recombinant) human growth factors are added: PDGF-BB, TGF-βand/or BMP-4. Additionally, one or several of the following (preferablyrecombinant) human growth factors can be used: EGF, aFGF, bFGF, IGF-1,SCF and SCGF. The use of EGF, PDGF-BB, IGF-1 and bFGF in combinationwith BMP-4 represents a particularly preferred embodiment of theinvention.

The induction of the differentiation of the multipotent stem cells intothe neuronal cell lineage, i.e. into neuronal progenitor cells and intomature neuronal cells is achieved by using the following (preferablyrecombinant) human growth factors: NGF, CNTF, GDNF and/or BDNF.Additionally one or several of the following (preferably recombinant)human growth factor can be used: EGF, bFGF, IGF-1, IL-1b, Il-6, Il-11,LIF, Flt3 ligand, SCF and BMP-4. The use of BDNF, GDNF, EGF plus bFGFrepresents an embodiment preferred according to the invention.

In order to induce a hepatocytic differentiation, the (preferablyrecombinant) human growth factor HGF is added. Additionally, one orseveral of the following (preferably recombinant) human growth factorscan be used: EGF, IGF-1, insulin, HCG, KGF, TNF-α, Flt3 ligand, SCF andSCGF. The use of Flt3 ligand, SCF, HGF plus TGF-β represents aparticularly preferred embodiment of the invention.

In order to characterise the development stage of the cells in theculture, it is necessary to check the differentiation phase lastingapproximately 10 to 14 days at regular intervals. A functionalexamination of the cells in the culture, e.g. in the form of a colonyassay, is suitable, for example. During the differentiation of themultipotent stem cells into the endothelial cell lineage, the EPCs loosethe ability to form blood cell colonies, e.g. with an increasingdifferentiation. By removing cell samples in phase II, it is thuspossible to check at regular intervals of e.g. 1 to 3 days whether andto what extent the ability of the cells to form colonies of the celllineage not desired in each case has changed. As soon as the cells formonly colonies of the desired cell lineage, the differentiation phase hasreached the stage in which only progenitor cells of this cell lineageare present. The cells can either be removed and/or isolated for furtherapplications or differentiated into mature cells of the desired celllineage.

In addition to or instead of the colony assay, the cells can be examinedin phase II by means of immunocytochemistry in order to verify theformation of certain surface structures on the cells during thedifferentiation phase. The results of the functional assay can becompared in an advantageous manner with those of the immunocytochemicalanalyses in order to find out which surface structures need to be formedif progenitor cells of the desired cell lineage are present, i.e. thecells have not yet matured but nevertheless lost the ability of theother cell lineages to form colonies.

The progenitor cells isolated in the manner described above must be usedeither immediately in the desired manner, i.e. for the plannedapplication, or be frozen. For endothelial progenitor cells, a mediumconsisting of DMSO, IMDM and HSA (preferably 40% IMDM+50% HSA+10% DMSO)has proved advantageous.

The present invention allows the use of ex vivo expanded multipotentstem cells and of hematopoietic, endothelial and mesenchymal progenitorcells and mature cells for the diagnosis, prophylaxis and therapy ofcardiovascular and malignant diseases. In addition, the multipotent stemcells, endothelial progenitor cells and mature endothelial cellsexpanded ex vivo can be used for coating surfaces. The multipotent stemcells expanded ex vivo and the endothelial and mesenchymal progenitorcells and mature cells can also be used for tissue engineering of organsand tissues.

In the following, a few practical examples of cells expanded anddifferentiated in vitro are provided as examples, the uses mentionedhaving the purpose merely of clarifying the application possibilitiesfor the multipotent stem cells according to the invention duringexpansion and/or differentiation, without restricting the inventionthereto.

Application I: Transplantation of Multipotent Stem Cells forHematopoietic Differentiation In Vivo

The multipotent stem cells expanded ex vivo can be used for allogenic orautologous transplantation in patients who, due to a malignant disease,are treated by myeloablative chemotherapy in order to regenerate bloodformation. In this case, the growth factor G-CSF is first administeredto the patients in order to effect a mobilisation of the bone marrowstem cells into the peripheral blood. Instead of leukaphereses, bloodcan be taken from the patients in the normal manner. From the peripheralblood, the stem cells are then isolated and the quantity of stem cellsrequired for a transplant generated by ex vivo expansion. It is thuspossible to avoid subjecting the patients to stresses and risksconnected with the execution of leukaphereses. On the one hand, thetransplant may consist exclusively of multipotent stem cells expanded exvivo. Alternatively, a transplant can be used which consists of expandedstem cells and endothelial progenitor cells. By the additional use ofthe endothelial progenitor cells, the reconstitution of the bone marrowfunction of the patients can be accelerated.

Application II: Transplantation of Genetically Modified Stem Cells andProgenitor Cells

Since the expansion method according to the invention and/or phase I ofthe two-stage method is a phase in which the multipotent stem cellsproliferate strongly, a simultaneous gene transfection can be carriedout advantageously. Corresponding methods for gene transfection byvectors, liposomes, naked DNA or electroporation are well known to theperson skilled in the art (compare “References”). In this connection, itmay be advantageous to use a vector for gene transfection which allowsan expression of the foreign gene inducible externally, i.e. of the genenot naturally expressed in the cells, such as e.g. the so called“Tet-on-system”, consisting of a transactivator construct and aresponder construct (compare Gossen M., Bujard H. Proc. Natl. Acad. Sci.89, 5547-5551, 1992; Gossen et al., Science 268, 1766-1769, 1995; Puttinet al., Am. J. Physiol. Renal Physiol. 281, F1164-72, 2001).

The stem cells expanded ex vivo and the endothelial progenitor cells canthus be genetically modified before transplantation and used fordiagnostic and therapeutic applications in the case of malignant tumoursand leukaemia. It is, for example, possible to modify the multipotentstem cells and endothelial progenitor cells expanded ex vivo geneticallyin such a way that they inhibit angiogenesis. This can be achieved e.g.by introducing a gene which encodes an angiogenesis inhibitingsubstance. The angiogenesis inhibiting substances comprise e.g.endostatin or angiostatin as well as antibodies or antisense nucleicacids against angiogenic cytokines, such as e.g. VEGF. Another possibleapplication is gene therapy of hereditary diseases such as for examplehemophilia A and B (compare Mannuci P M, Tuddenham E G. N. Engl. J. Med.344, 1773-1779, 2001; Emilien et al., Clin. Lab. Hematol. 22, 313-322,2000), Gaucher's disease (compare Barranger et al., Baillieres Clin.Hematol. 10, 765-768, 1997), glycogenosis (type I-III) (compare ElpelegO N. J. Pediatr. Endocrinol. Metab. 12, 363-379, 1999),mucopolysaccharidosis (type I-VII) (compare Caillaud C, Poenaru L.Biomed. Pharmacother. 54, 505-512, 2000), Niemann-Pick disease (compareMillat et al., Am. J. Hum. Genet. 69, 1013-1021, 2001; Miranda et al.,Gene Ther. 7, 1768-1776, 2001), Hirschsprung's disease (compare Amiel etal., J. Med. Genet. 38, 729-739, 2001), Fanconi anemia (compareYamashita T. Int. J. Hematol. 74, 33-41, 2001), Chediak-Higashi syndrome(compare Ward et al., Traffic 1, 816-822, 2000), thalassemia (compareWeatherhall D J. Nat. Rev. Genet. 2, 245-255, 2001), sickle cell anaemia(compare Chui D H, Dover G J. Curr. Opin. Pediatr. 13, 22-27, 2001) etc.

Methods for gene transfection suitable within the framework of thepresent invention are described in the publications quoted in the“References” section, these processes being mentioned only as exampleswithout being limited to these methods.

Application III: Diagnosis of Metastases and Ischemic Diseases

III a.) Diagnosis of Metastases

As a special application in oncology, the ex vivo-expanded multipotentstem cells and/or the endothelial progenitor cells can be radioactivelylabelled with 18F fluorodeoxyglucose (¹⁸F-FDG) or with ¹¹¹indium andadministered to patients intravenously in order to visualise metastases.The administered cells are built up in the tumour tissue (compare deBont et al., Cancer Research 61, 7654-7659, 2001), as a result of whichthe metastases can be visualised by diagnostic routine processes such aspositron emission tomography (PET, for the detection of ¹⁸F-FDG labelledcells) and/or simple scintigraphy (for the detection of¹¹¹indium-labelled cells).

III b.) Diagnosis of Ischemic Lesions

The radioactive labelling of ex vivo-expanded multipotent stem cellsand/or the endothelial progenitor cells with 18F-fluorodeoxyglucose(¹⁸F-FDG) or with ¹¹¹indium can also be used for the diagnosis ofischemic diseases. Following intravenous administration, the labelledcells migrate via the circulation into ischemic regions of the organismin order to participate there in angioneoplasm (compare survey by Masudaet al., Hum. Cell 13, 153-160, 2000). In this way, it is possible todetect also undervascularizations which are clinically without symptoms.The visualisation of the labelled cells takes place by PET and/orscintigraphy analogous to the above-mentioned process.

Application IV: Angioneoplasm In Vivo

The ex vivo-expanded multipotent stem cells and the endothelialprogenitor cells and mature endothelial cells can also be used for thetherapy of diseases involving a reduced vascular supply. It is, forexample, possible to introduce the ex vivo-expanded multipotent stemcells, the endothelial progenitor cells or the mature endothelial cellsdirectly into an organ and/or vessel system in order to induce theformation of new blood vessels therein. The reduced vascular supply canbe due to an ischemic disease or an autoimmune disease. Affected tissuescan comprise muscles, the brain, kidneys, lung. The ischemic tissues canconsist particularly of a myocardial ischemia, ischemic myocardiopathy,renal ischemia, pulmonal ischemia or an ischemia of the extremities. Theex vivo-expanded stem cells and the endothelial progenitor cells can begenetically modified before introduction into the diseased organ and/orvessel in order to increase the therapeutic effect. It is, for example,possible to transfect the ex vivo-expanded stem cells and endothelialprogenitor cells with a gene which encodes a vasodilatory substance.

Application V: Reendothelialisation of Vessels

As a special application in cardiology, both the ex vivo-expanded stemcells and the endothelial progenitor cells and mature endothelial cellscan be used for the treatment of diseases and injuries of the coronaryarteries. It is, for example, possible to apply the multipotent stemcells or the endothelial progenitor cells following an angioplasty orrotablation directly intracoronal in order to acceleratereendothelialisation of the injured coronary sections, thus preventingrestenosis. This application can also be transferred to the treatment ofdiseases and injuries of arteries in other localities such as e.g.vessels of the extremities, by injecting the expanded stem cells, theendothelial progenitor cells or the mature endothelial cells directlyinto the vessel concerned.

Application VI: Coating of Coronary Stents

Moreover, the endothelial progenitor cells and mature endothelial cellsobtained by the differentiation of the multipotent stem cells can beused for coating coronary stents which are implanted followingangioplasty or rotablation in order to prevent restenosis. In this case,the endothelial progenitor cells or the mature endothelial cells can beapplied either directly onto the stent surface or on matrix-coatedstents.

Different stent surfaces can be used: ceramics, PTFE, gold, titaniumetc. The matrix can consist e.g. of fibronectin, collagen, heparin,gelatine, fibrin, silicone, phosphoryl choline or matrigel.Additionally, the matrix can be coupled with antibodies bindingendothelial cell-specific or progenitor cell-specific surface antigens.The following antibodies can be used: anti-CD7 mAb, anti-CD31-mAb,anti-CD34 mAb, anti-CD54 (ICAM-1) mAb, anti-CD62e mAb (E-selection),anti-CD90 (Thy-1) mAb, anti-CD106 mAb (VCAM-1), anti-CD114 (G-CSF-R)mAb, anti-CD116 (GM-CSF-R) mAb, anti-CD117 (c-kit) mAb, anti-CDw123(IL-3R α chain) mAb, anti-CD127 (IL-7R) mAb, anti-AC133 mAb, anti-CD135(Flk3/Flk2) mAb, anti-CD140b (PDGF-RP) mAb, anti-CD144 (VE-cadherin)mAb, anti-CD164 mAb, anti-CD172a mAb, anti-CD173 mAb, anti-CD174 mAb,anti-CD175 mAb, anti-CD176 mAb, anti-CD184 (CXCR4) mAb, anti-CD201(endothelial cell protein C receptor) mAb, anti-CD202b (Tie-2/Tek) mAb,anti-CD224 mAb, anti-CD227 (MUC-1) mAb, anti-CD228 mAb, anti-CD243(MDR-1) mAb, anti-EGF-R mAb, anti-FGF-R mAb, anti-P1H12 mAb, anti-KDRmAb, anti-BENE mAb antibodies against lectins. The endothelialprogenitor cells can be used for the coating in a genetically unmodifiedor gene transfected state. For the transfection, genes encoding avasodilatory substance such as e.g. NO synthase or genes encoding anantithrombotic substance such as e.g. antithrombin III can be used.

Application VII: Coating of Vascular Valves

A further use of the endothelial progenitor cells and mature endothelialcells obtained in culture consists of the coating of biomechanicalvascular valves of the heart in order to prevent thrombosation ofimplanted vascular valves.

According to the two above-mentioned practical examples, the inventionalso relates to methods for coating implantable materials, in particularcoronary stents and vascular valves, in the case of which the two-stageexpansion/differentiation method according to the invention is carriedout and, in the case of endothelial differentiation during phase IIand/or at the end of phase II (depending on whether a coating with EPCsand/or mature ECs is desired), the material to be implanted, which ispreferably coated with fibronectin, is transferred into the culturemedium in which the differentiation of the cells takes place. Accordingto a preferred embodiment of the invention, the stem cells can be genetransfected in phase I such that coating takes place withgene-transfected EPCs and/or ECs.

Application VIII: Tissue Engineering of Organs

A possible field of application for the ex vivo-expanded multipotentstem cells and for the endothelial and mesenchymal progenitor cells istissue engineering. The ex vivo-expanded multipotent stem cells can beused in order to produce organ-specific tissues such as brain, liver,heart, cartilage, bone, retinal, muscle or connective tissue in vitro.For this purpose, the stem cells are cultivated in special basal media.To generate neuronal cells, the media SATO medium or DMEM-F12, forexample, can be used. For the preparation of liver cells, media such ase.g. Williams medium E can be used. The cultures can contain additionsof serum. Alternatively, serum-free culture systems can be used.

For the induction of the neuronal differentiation, the multipotent stemcells can be cultivated in the presence of at least one growth factorfrom the group consisting of NGF, ciliary neurotrophic factor (CNTF),GDNF and BDNF and optionally in combination with at least one growthfactor from the group consisting of EGF, bFGF, IGF-1, IL-1b, IL-6,IL-11, LIF, Flt3 ligand, SCF and SCGF.

For tissue engineering of liver cells, the multipotent stem cells can becultivated in the presence of HGF and, optionally, in combination withat least one growth factor from the group consisting of EGF, IGF-1,insulin, HCC, keratinocyte growth factor, TNF-α, TGF-β, Flt3 ligand, SCFand SCGF.

In connection with the tissue engineering of organs and tissues, thefollowing methods can be used: liver (compare Torok et al., Dig. Surg.18, 196-203, 2001), brain (compare Woerly S. Neurosurg. Rev. 23, 59-77,2000; Tresco P A. Prog. Brain Res. 128, 349-363, 2001), heart (compareMann B K, West J L. Anat. Rec. 263, 367-371, 2001), cartilage (compareLaurencin et al., Annu. Rev. Biomed. Eng. 1, 19-46, 1999; Lu et al.,Clin. Orthop. 391, S251-270; Gao et al., Tissue Eng. 7, 363-371, 2001),bone (compare Doll et al., Crit. Rev. Eukaryot. Gene Expr. 11, 173-198,2001; Gao et al., Tissue Eng. 7, 363-371, 2001), retina (compare Lu etal., Biomaterials 22, 3345-55, 2001), muscle tissue (compare Polinkovicet al., Crit. Rev. Eukaryot. Gene Expr. 11, 121-129, 2001), connectivetissue (compare Pieper et al., Biomaterials 21, 1689-1699, 2001), skin(Houzelstein et al., Development 127, 2155-64, 2000; Ng et al., TissueEng. 7, 441-455, 2001), kidney (compare Amiel et al., World J. Urol. 18,71-79, 2000; Poulson et al., J. Pathol. 195, 229-235, 2001). Whenestablishing the vessel supply in organs prepared by tissue engineering,the method of Kaihara et al. (Tissue Eng. 6, 105-117, 2000) can be used.

To produce artificial tissues, in particular brain, liver, kidney,heart, bone, retinal, muscle or connective tissue or skin, a matrix canbe provided which is brought into contact with the multipotent stemcells, progenitor cells and/or differentiated cells expanded accordingto the invention. This means that this matrix is transferred into asuitable vessel and a layer of the cell-containing culture medium isplaced on top (before or during the differentiation of the expandedmultipotent stem cells). The term “matrix” should be understood in thisconnection to mean any suitable carrier material to which the cells areable to attach themselves or adhere in order to form the correspondingcell composite, i.e. the artificial tissue. Preferably, the matrix orcarrier material, respectively, is present already in athree-dimensional form desired for later application. According to aparticular embodiment of the invention, bovine pericardial tissue isused as matrix which is crosslinked with collagen, decellularised andphotofixed (CardioFix™, Sulzer Medica, Forich, Switzerland).

Application IX: Tissue Engineering of Blood Vessels

Moreover, the ex vivo expanded multipotent stem cells and theendothelial progenitor cells and mature endothelial cells can be usedfor the in vitro preparation of blood vessels. The blood vesselsgenerated in vitro can be implanted as vascular transplants in patientswith coronary heart disease or peripheral arterial vascular occlusionsand represent an alternative to the bypass operation and to implantingof artificial vessel prostheses.

Regarding the process for the production of artificial blood vessels,reference is made to the details provided regarding the production ofartificial tissue, in particular regarding the “matrix” used, in orderto avoid repetition. The matrix is preferably preformed in the form of acylinder.

Application X: Angioneoplasm in Organ Transplants and Tissue Transplants

The ex vivo-expanded multipotent stem cells and the endothelialprogenitor cells can also be used to improve or to guarantee thevascular supply for skin transplants. The skin transplants can comprisemesh grafts or skin transplants produced by tissue engineering.

In addition, the ex vivo expanded multipotent stem cells and theendothelial progenitor cells can be used in order to guarantee avascular supply for organs or tissues produced by tissue engineering.The organs or tissues can comprise e.g. liver, kidney or cartilage. Byusing autologous multipotent stem cells or autologous endothelialprogenitor cells, vascular systems can be produced individually for thepatient in order to possibly prevent a host versus transplant reaction(transplant rejection).

With respect to the above-mentioned uses, a further object of thepresent invention consequently consists of a method for the productionof a pharmaceutical composition in which the method according to theinvention for the expansion of multipotent stem cells is carried out.Insofar as additionally to or instead of the stem cells, progenitorcells (e.g. endothelial progenitor cells) and/or matured cells (e.g.mature endothelial cells) are to be used, the differentiation phase mayfollow according to the invention such that the two-stageexpansion/differentiation method is carried out for the preparation ofthe pharmaceutical composition, the cells being isolated during and/orat the end of phase II, depending on the desired degree ofdifferentiation. The cells obtained in each case can be used directlyfor therapy, preferably by being taken up in 0.9% saline solution or,insofar as required, processed in another way for the administrationconcerned. If necessary, this includes radioactive labelling of thecells.

According to a particular embodiment of the invention, thepharmaceutical composition may contain a mixture of expanded multipotentstem cells and endothelial progenitor cells. The process for thepreparation of the pharmaceutical composition consequently includes, ifnecessary, the execution of the expansion/differentiation methodaccording to the invention (i.e. phase I and II), wherein cells obtainedin phase I are combined with EPCs isolated in phase II.

According to a further embodiment of the invention, the process for theproduction of a pharmaceutical composition can also include a genetransfection, i.e. the introduction of foreign genes into themultipotent stem cells, the transfection taking place within theframework of the expansion process (e.g. during the two-stage processduring the expansion phase, phase I). Insofar as the geneticallymodified multipotent stem cells are further differentiated, it is alsopossible to provide a pharmaceutical composition which contains bothgene transfected stem cells and gene transfected progenitor cells.

According to the invention, the term “pharmaceutical composition”includes both preparations for therapeutic application and agents fordiagnostic purposes.

Moreover, the invention relates to the use of the cells obtained by theexpansion process according to the invention and of the cells obtainedby the two-stage expansion/differentiation process according to theinvention (i.e. multipotent stem cells, progenitor cells and maturedcells) for the production of artificial organs and tissues, inparticular of brain, liver, kidney, heart, cartilage, bone, retinal,muscle or connective tissue or skin.

The subject matter of the invention moreover consists of pharmaceuticalcompositions, implantable materials and artificial organs and tissue, inparticular including the blood vessels, produced by using the expandedmultipotent stem cells, progenitor cells and/or mature cells producedaccording to the invention (and/or obtainable by using a processaccording to the invention).

ADVANTAGES OF THE INVENTION

The present invention describes a culture system which allows an ex vivoexpansion of multipotent human stem cells. In comparison with culturesystems previously described, the present invention has the advantagethat no or no major differentiation of the stem cells occurs during theexpansion phase. As a result, the stem cells retain their regenerativecapacity and can be used for autologous or allogeneic transplants inpatients with malignant diseases. Moreover, they can be used for tissueengineering. The invention is moreover characterised in that themultipotent stem cells can be gene transfected under the cultureconditions developed. As a result, new approaches for the diagnosis andtherapy of cardiovascular and malignant diseases are obtained. Finally,the invention makes it possible that endothelial progenitor cells aremultiplied a hundred fold in the culture system and consequently cellcounts are reached such as they are necessary for clinical applications.In this respect, the culture system has the advantage that both themultipotent stem cells and the endothelial progenitor cells can beproduced without major expenditure on equipment.

Important applications of the stem cells expanded and/or differentiatedaccording to the invention are summarised in the following as anexample:

-   -   Transplantation with ex vivo expanded multipotent stem cells    -   Transplantation with ex vivo expanded multipotent stem        cells+endothelial progenitor cells    -   Transplantation with genetically modified multipotent stem cells        with the following transgene:        -   angiogenic inhibiting gene        -   angioneoplasm promoting gene    -   Transplantation with genetically modified multipotent stem cells        for the therapy of hereditary diseases    -   Diagnosis of metastases    -   Diagnosis of ischemia    -   Angioneoplasm in vivo    -   Reendothelialisation of vessels in vivo    -   Tissue engineering of brain, liver, kidney, heart, cartilage,        bone, retinal, muscle or connective tissue or of skin    -   Production of artificial blood vessels    -   Vascular supply for skin transplants    -   Vascular supply for organs or tissue produced artificially (by        tissue engineering).

In the following, the invention will be described by way of examples.

EXAMPLES

The substances, growth factors and antibodies mentioned above and in thefollowing examples are either commercially available or they can beproduced and/or obtained according to known methods. A survey of therelevant publications are given in the appendix under “Reference”.

Example 1

Sample Preparation:

For this example, a leukapheresis product, kept under cryogenicconditions, of a patient was used who, as a result of a malignantdisease, had been assigned to high dosage chemotherapy with autologousstem cell transplantation. However, fresh leukapheresis products orG-CSF mobilised, non-pheresised blood can also be processed. The samplekept under cryogenic conditions was defrosted in a first step at 37° C.in a water bath and transferred into a buffer consisting of PBS, 0.5%HSA and 0.6% ACD-A. The sample was then centrifuged for 15 minutes at900 rpm and 4° C. The cell pellet obtained was resuspended in PBS+5%HSA. Subsequently, DNAse (100 U/ml) was added to this PBS solution andthe sample was incubated for 30 minutes on an automatic mixer.

Fresh leukapheresis product and peripheral, non-pheresised blood can bepassed directly to density gradient centrifugation.

Immunomagnetic AC133 Selection:

By density gradient centrifugation via Fikoll-Hypaque, the mononuclearcell fraction (MNC) of the leukapheresis product was obtained. For thispurpose, the sample was centrifuged for 20 minutes at 2000 rpm and 4° C.Subsequently, the sample was washed twice for 10 minutes at 1200 rpm inPBS+0.5% HSA+DNAse (100 U/ml). The MNC were then resuspended in PBS+0.5%HSA, incubated with AC133-conjugated microbeads (AC133 isolation kit,Miltenyi Biotec, Bergisch-Gladbach) for 30 minutes at 4° C. and washedin PBS+0.5% HSA for 10 minutes at 1200 rpm. The AC133 selection was thencarried out on the autoMACS (Miltenyi Biotec; Posseldx softwareprogram). Following each selection, the degree of purity was determinedby FACS analysis.

Suspension Cultures:

The freshly isolated AC133⁺ cells were cultivated in fibronectin-coatedplates with 24 well plates at a cell density of 2×10⁶ cells/ml inIMDM+10% FCS+10% horse serum+10⁻⁶ mole/l hydrocortisone. For theexpansion of the AC133⁺ cells, the following recombinant human growthfactors were added to the medium: SCGF (100 ng/ml; TEBU, Frankfurt),Flt3 ligand (50 ng/ml; TEBU) and VEGF (50 ng/ml; TEBU) and the cellswere incubated for 14 days at 37° C. in 5% CO₂. For the differentiationof the stem cells, the medium was supplemented with SCGF (100 ng/ml) andVEGF (50 ng/ml) and the cells were cultivated for 14 days. Additionalfeeding of the cultures was carried out depending on the proliferationof the cells. In this case, the supernatant was removed carefully with apipette and replaced by fresh medium. The proliferating cells containedin the supernatant were counted, adjusted to a cell density of 2×10⁶cells/ml and introduced into fresh wells of the well plate.

Colony Assays:

Freshly isolated AC133⁺ cells and cells which had been expanded for 8and 14 days were introduced with a cell density of 1×10³ to 5×10⁴ into asemisolid medium which consisted of 0.9% methylcellulose in IMDM, 30%FCS, 1% calf serum albumin, 10⁻⁴ mol/l mercaptoethanol and 2 mmol/lL-glutamine (complete medium from Cell Systems, St. Katharinen). Inparallel batches, the cultures were stimulated either with a combinationof hematopoietic growth factors consisting of SCF (50 ng/ml), IL-3 (20ng/ml), IL-6 (20 ng/ml), G-CSF (20 ng/ml), GM-CSF (20 ng/ml) anderythropoetin (3 U/ml; complete medium from Cell Systems) or with acombination of SCGF (100 ng/ml, TEBU) and VEGF (50 ng/ml, TEBU). Allcultures were carried out in quadruplicate, incubated at 37° C. in 5%CO₂ and evaluated after 14 days under the inversion microscope.

Immune Staining:

Freshly isolated AC133⁺ cells and cultivated cells were centrifuged in acytocentrifuge at 500 rpm for 5 minutes on slides. The cytospins wereair dried for at least 24 hours and then stained by immunofluorescence.The following primary non-conjugated and conjugated antibodies wereused: anti-KDR-mAb (Sigma), anti-Ulex Europaeus agglutinin-1 mAb,anti-EN4 (cell systems), anti-CD31-PE (Pharmingen, Hamburg),VE-cadherin-PE (Pharmingen) and anti-vWF-FITC. Anti-mouseFITC-conjugated immunoglobulins were used as secondary antibodies. Thecytospins were first washed in 10% FCS/PBS in order to blocknon-specific binding sites. Subsequently, the cytospins were incubatedfor 60 minutes at room temperature with the primary antibody. Thecytospins which were incubated with a non-conjugated primary antibodywere subsequently incubated for 30 minutes at room temperature.Subsequently, the cytospins were fixed with 5% glacial aceticacid/ethanol at −20° C. for 15 minutes.

Flow Cytometry:

The freshly isolated AC133+ cells were first incubated with a hemolyticbuffer (0.155 mol/l NH₄Cl, 0.012 mol/l NaHCO₃, 0.1 mmol/l EDTA, pH 7.2)in order to lyse the erythrocytes. Cells which had already beencultivated were passed directly to antibody incubation. 5×10⁵ cells wereincubated in each case with the following antibodies: PE-anti-AC133 mAb,FITC-anti-CD34 mAb, PE-anti-CD33 mAb, FITC-anti-CD105 mAb, PE-anti-CD14mAb, FITC-anti-CD45 mAb, PE-anti-VE-cadherin mAb, FITC-anti-vWF mAb,PE-anti-CD31 mAb, PE-anti-c-kit mAb, FITC-anti-CD90 mAb and PE-anti-CD7mAb. All incubations were carried out for 15 minutes at 4° C.Subsequently, the cells were washed in 0.1% BSA/PBS. The measurementswere carried out as single colour and two colour analyses on a FACS SCANflow cytometer (Becton Dickinson) and with the Cell Quest softwareprogram. Each analysis included at least 5000 counting events. Anisotype control (γ1γ2a, Parmingen) was carried out concurrently witheach measurement.

Synthesis of cDNA and Reverse Transcription Polymerase Chain Reaction(RT-PCR):

Freshly isolated AC133⁺ cells and cultivated cells were washed twice inPBS and centrifuged for 5 minutes at 1200 rpm and room temperature. Theisolation of the RNA was carried out by means of a mini-column (RneasyKit, Quiagen, Hilden) in line with the manufacturer's instruction. Onemicrogram of the isolated RNA was used for the cDNA synthesis. The cDNAsynthesis was carried out using the avian myeloblastosis virus (AMV)reverse transcriptase and oligo dT as primer. Different aliquots of thecDNA were amplified by means of specific primers for KDR, Tie-2/Tek,VE-cadherin and vWF and for actin as positive control. For KDR,Tie-2/Tek, Ve-cadherin and vWF, two passages, and for actin one passagewas carried out with 35 cycles of the PCR in a programmable thermoblockat 94° C. for 1.5 minutes, at 60° C. for 3 minutes and at 72° C. for 4minutes. The PCR products were separated on 1% agarose gel, stained withethidium bromide and visualised under UV light. The primer sequenceswere as follows: outer KDR sense primer 5′-GTCAAGGGAAAGACTACGTTGG-3′,outer KDR antisense primer 5′-AGCAGTCCAGCATGGTCTG-3′, inner KDR senseprimer 5′-CAGCTTCCAAGTGGCTAAGG-3′, inner KDR antisense primer5′-TCAAAAATTGTTTCTGGGGC-3′, outer Tie-2/Tek sense primer5′-TGGACCTGTGAGACGTTC-3′, outer Tie-2/Tek antisense primer5′-CTCTAAATTTGACCTGGCAACC-3′, inner Tie-2/Tek sense primer5′-AGGCCAACAGCACAGTCAG-3′, inner Tie-2/Tek antisense primer5′-GAATGTCACTAAGGGTCCAAGG-3′, outer VE-cadherin sense primer5′-DAYCATTGGATACTCCATCCG-3′, outer VE-cadherin antisense primer5′-ATGACCACGGGDAYGAAGTG-3′, inner VE-cadherin sense primer5′-TTCCGAGTCACAAAAAAGGG-3′, inner VE-cadherin antisense primer5′-TATCGTGATTATCCGTGAGGG-3′, outer vWF sense primer5′-CTGCAAGGTCAATGAGAGAGAGG-3′, outer vWF antisense primer5′-GAGAGCAGCAGGAGCACTG-3′, inner vWF sense primer5′-TGAGGAGCCTGAGTGCAAC-3′, inner vWF antisense primer5′-TGGAGTACATGGCTTTGCTG′. The specific primers for KDR, Tie-2/Tek,VE-cadherin, vWF and actin recognise encoding sequences. The size of thePCR products was as follows: for the outer KDR primer pair 591 bp, forthe inner KDR primer pair 213 bp, for the outer Tie-2/Tek primer pair624 bp, for the inner Tie-2/Tek primer pair 323 bp, for the outerVE-cadherin primer pair 462 bp, for the inner VE-cadherin primer pair340 bp, for the outer vWF primer pair 312 bp, for the inner vWF primerpair 128 bp. In order to avoid cross-contamination, the individualoperating steps of the PCR reaction and gel electrophoresis were carriedout in different rooms using different pipettes. Accordingly, controlreactions carried out simultaneously were always negative.

Results of Example 1:

Characterisation of the Freshly Isolated AC133⁺ Cell Population

The flow cytometric analyses gave a degree of purity of 99.94%. Thetotal population of the AC133+cells coexpressed the surface antigensCD34, CD45, CD33 and CD31. 42.3% of the AC133⁺ cells coexpressed CD90(thy-1), a surface marker which is expressed only on very non-maturestem cells. CD7 and c-kit, also markers for non-mature stem cells, wereexpressed by 15.23% and 6.86% of the AC133⁺ cells. The endothelial cellmarkers vWF and VE-cadherin were detectable only on 1.43% and 0.36% ofthe cells.

Proliferation and Differentiation of the AC133+ Cells in SuspensionCulture:

Initially, the AC133⁺ cells were expanded for 14 days under theinfluence of Flt3 ligand, SCGF and VEGF. The cells were adherent afteronly a few hours following the beginning of the culture. During thefirst four culture days, the cells formed a monolayer of small roundcells. The cell density increased substantially every day. On day 5 ofthe culture period, a non-adherent cell layer of small round cells wasthen obtained which had formed above the adherent cell layer. Thenon-adherent cell layer was carefully removed with a pipette, countedand introduced into fresh wells of the well plate. It was then possibleto repeat the process, the cells proliferated continuously. On day 14 ofthe culture period, a 100 fold multiplication of the cells was achieved.The morphology changed only slightly during the entire period. On day14, the cells had a larger diameter and exhibited a “cobblestone”morphology. On day 14, the cells were transferred into a medium whichcontained the growth factors SCGF and VEGF. Within three to four days,the proliferation subsided substantially and the cells exhibited thefirst morphological differentiation characteristics typical ofendothelial cells. Initially, small elongate cells were obtained whichgrew while remaining very flat. After a 14 day culture in thedifferentiation medium, the cell population consisted predominantly oflarge spindle-shaped cells with a typical endothelial cell morphology.

Clonogenic Potential of the Freshly Isolated AC133⁺ Cells and the Cellsin Culture:

The freshly isolated AC133⁺ cells and cells expanded for 14 days were,introduced into a semi-solid medium which contained either hematopoieticgrowth factors for purposes of the stimulation of hematopoietic coloniesor the cytokines SCGF and VEGF for purposes of the induction ofendothelial colonies. As shown in Table 1, the cells which had beenexpanded for 14 days in suspension cultures still exhibited a clonogenicpotential. In comparison with freshly isolated AC133⁺ cells, these cellswere no longer capable of forming BFU-E and CFU-E but instead had ahigher capacity for forming endothelial colonies. TABLE 1 Clonogenicpotential of the freshly isolated AC133+ cells and of the cultivatedcells. BFU-E CFU-E CFU-GEMM CFU-GM CFU-G CFU-M CFU-EC AC133+ 17 77 0 252104 20 4 day 0 Expand. 0 0 0 36.5 6 24 33 day 14Abbreviations: BFU-E = burst-forming unit erythrocyte; CFU-E =colony-forming unit erythrocyte; CFU-GEMM = colony-forming unitgranulocyte-erythrocyte-macrophage-megakaryocyte; CFU-GM =colony-forming unit granulocyte-macrophage; CFU-G = colony-forming unitgranulocyte; CFU-M = colony-forming unit macrophage; CFU-EC =colony-forming unit endothelial cell.Identification of the Endothelial Cells:

In order to identify cells of the endothelial cell lineage, theexpression of the endothelial cell markers CD31, vWF, VE-cadherin, Ulexeuropaeus agglutinin-1, Tie-2/Tek and KDR were examined by immunestaining and RT-PCR. The results are given in Table 2 and Table 3. TABLE2 Percentage of positive cells for CD31, vWF, VE-cadherin and Ulexeuropaeus agglutinin-1 by immunofluorescence staining. CD31 vWFVE-cadherin Ulex AC133⁺ cells 99% 0 0 0 day 0 expanded cells 99% 5% 0 0day 14 Differentiated 99% 99%  99% 99% cells day 14

TABLE 3 Gene expression analysis of the freshly isolated AC133+ cellsand the cultivated cells by RT-PCR. KDR Tie-2/Tek vWF VE-cadherin AC133⁺cells Negative Negative Positive Negative day 0 Expanded cells PositivePositive Positive Negative day 14 Differentiated Positive PositivePositive Positive cells day 14

Example 2

In Vivo Studies with Gene Transfected AC133⁺ Cells

In this example, the AC133⁺ cells were cultivated for 4 days at a celldensity of 2×10⁶ cells/ml in IMDM+10% FCS+10% horse serum+10⁻⁶ mol/lhydrocortisone+Flt3 ligand (50 ng/ml)+SCGF (100 ng/ml)+VEGF (50 ng/ml).On day 5, 6 and 7 of the culture period, the AC133+ cells weretransfected with the retroviral vector SF11αEGFPrev which encodes theenhanced green fluorescent protein. For this purpose, 6-well plates werefirst coated with the recombinant fibronectin fragment CH296 (RN,compare e.g. R. Kapur et al., Blood 97 (2001) 1975-81; Takara, Otsu,Japan). Subsequently, 2.5 ml/well of the retroviral supernatantpreviously obtained in cultures of the cell lineage PG13 werecentrifuged for 30 minutes at 1000 g and 4° C. onto the RN-coated wellplates. In order to load the well plates with retroviral particles tothe maximum, the centrifuging process was repeated four times. Then, theAC133⁺ cells were introduced into the RN-coated well plates loaded withvirus particles and incubated overnight at a cell density of 2×10⁶cells/ml in the culture medium described above. The transfection processwas repeated on the following 4 consecutive days. For this purpose, afresh 6-well plate was coated with RN at the beginning of transfectionin each case and loaded with retroviral particles in 5 centrifugingsteps, as described above, and the transfection was carried outovernight. In this way, a transduction efficiency of 70% was achieved.The transfected cells were then cultivated in 6-well plates coated withfresh fibronectin, which had not been loaded with viral particles, for afurther 48 hours in the above-mentioned medium under the influence ofFlt3 ligand, SCGF and VEGF. On day 9 of the culture period, the cellswere trypsinised, washed, resuspended in 100 μl of PBS/1×10⁶ cells andsubcutaneously injected into SCID mice. In this connection, threedifferent test groups of ten mice each were formed. In group I, asuspension of 1×10⁶ gene transfected cells plus 1×10⁶ cells of the lungcarcinoma cell line A549 was injected into each mouse. The test animalsof group II received 1×10⁶ gene transfected cells exclusively whereas ingroup III, 1×10⁶ A549 cells were applied subcutaneously exclusively.Four weeks after the injection, the tumour size and structure in all thetest animals were analysed.

Subcutaneous tumours were present in all mice of group I and group III,whereas none of the animals in group II had formed a subcutaneoustumour. The largest tumours were detectable in the mice of group I. Inthis case, the tumour diameter was on average 30% greater than that ofthe tumours in group III. In frozen section preparations, the tumours ofgroup I moreover exhibited a greater vascular density than those ofgroup III. By fluorescence microscopy, the content of EGFP-expressingcells of the tumours was examined. Green fluorescent cells could bedetected in the vessels in the case of tumours of group I.

Example 3

In Vitro Differentiation of Liver Cells from AC133⁺ Cells

In this example, the AC133⁺ cells were cultivated at a cell density of2×10⁶ cells/ml in Williams medium E+10% FCS+10% horse serum+5×10⁻⁶ mol/lhydrocortisone+Flt3 ligand (50 ng/ml)+SCF (100 ng/ml)+HGF (50ng/ml)+TGF-β (10 ng/ml) in collagen-coated well plates. After 14 days,the cells were trypsinised and immunocytochemically analysed. 70% of thecells were positive for the hepatocytic marker OCH1E5 (DAKO). 20%expressed the biliar cell marker cytokeratin-19 (CK-19 (DAKO).

Example 4

In Vitro Differentiation of Neuronal Cells from AC133⁺ Cells

In this example, the AC133⁺ cells were cultivated at a cell density of2×10⁶ cells/ml in DMEM/F-12 (1:1)+5×10⁻³ mol/l hepes buffer+0.6%glucose+3×10⁻³ mol/l sodium bicarbonate+2×10⁻³ mol/l glutamine+25 μg/mlinsulin+100 μg/ml transferrin+50 ng/ml BDNF+50 ng/ml GDNF+EGF (20ng/ml)+bFGF (20 ng/ml) in uncoated well plates. After 14 days, the cellswere analysed immunocytochemically. 62% of the cells were positive forthe marker glial fibrillary acidic protein (GFAP; Incstar, Stillwater,Minn., USA), 7% of the cells expressed the microtubule-associatedprotein-2 (MAP-2; Boehringer Mannheim) and 3% of the cells were positivefor the oligodendrocyte marker O4 (Boehringer Mannheim).

Example 5

Stent Coating with Gene Transfected Endothelial Progenitor Cells

In this example, the AC133⁺ cells were cultivated for 4 days at a celldensity of 2×10⁶ cells/ml in IMDM+10% FCS+10% horse serum+10⁻⁶ mol/lhydrocortisone+Flt3 ligand (50 ng/ml)+SCGF (100 ng/ml)+VEGF (50 ng/ml)and subsequently gene transfected, as described above, with retroviralvector SF11αEGFPrev. Following renewed culture in IMDM, Flt3 ligand,SCGF and VEGF, the cells were trypsinised on day 9 of the cultureperiod, washed in PBS and taken up again in culture medium. In parallel,PTFE stents were coated for 2 hours with fibronectin. The coated stentswere then transferred into a centrifuge tube and a layer of 3 ml of thecell-containing culture medium was placed on top. The tubes thusprepared were then centrifuged for 2 hours at 12×g and 37° C.Subsequently, the coated stents were carefully transferred into a 25 cm²culture flask with 10 ml of the above-mentioned culture medium andanalysed at defined moments under the inversion fluorescence microscope.After 1 week, a confluent coating with fluorescent cells was stilldetectable on the stent.

Example 6

Coating of Biomechanical Vascular Valves of the Heart with GeneTranfected Endothelial Progenitor Cells

In a manner analogous to the above-mentioned example, the AC133⁺ cellswere first cultivated 4 days at a cell density of 2×10⁶ cells/ml inIMDM+10% FCS+10% horse serum+10⁻⁶ mol/l hydrocortisone+Flt3 ligand (50ng/ml)+SCGF (100 ng/ml)+VEGF (50 ng/ml) and subsequently genetransfected with retroviral vector SF11αEGFPrev, as described above. Thecells were then cultivated again in IMDM, Flt3 ligand, SCGF and VEGF andfrom day 15 of the culture period onwards in IMDM, SCGF and VEGF. On day28 of the culture period, the cells were trypsinised, washed in PBS andagain taken up in culture medium. In parallel, vascular valves werecoated for 2 hours with fibronectin and then introduced into a 75 cm²culture flask. In the horizontal position, 250 ml of the cell-containingculture medium were pipetted into the culture flask such that thevascular valve was completely surrounded by medium. The culture flaskswere then rotated slowly for 24 hours on an automatic mixer. Theautomatic mixer was placed in an incubator and the culture flasks wereincubated at 37° C. and 5% CO₂. Subsequently, the culture flasks wereremoved from the mixer, half of the culture medium was removed with apipette and replaced by fresh medium. The coating of the vascular valvewas analysed at defined moments under the inversion fluorescentmicroscope. In this example, too, a confluent layer of fluorescent cellswas still detectable after 1 week.

Example 7

Preparation of Blood Vessels from Endothelial Progenitor Cells In Vitro

In this example, the AC133⁺ cells were cultivated for 14 days at a celldensity of 2×10⁶ cells/ml in IMDM+10% FCS+10% horse serum+10⁻⁶ mol/lhydrocortisone+Flt3 ligand (50 ng/ml)+SCGF (100 ng/ml)+VEGF (50 ng/ml)and subsequently for a further 4 days under the influence of SCGF andVEGF. The cells were then trypsinised, washed in PBS and again taken upin culture medium. In parallel, a piece of bovine pericardial tissue,2×1 cm in length, which had been crosslinked with collagen,decellularised and photofixed (CardioFix™, Sulzer Medica, Zurich,Switzerland), was prepared and formed into a cylinder. These cylinderswere then transferred into a centrifuge tube and a layer of 3 ml of thecell-containing culture medium was placed on top. bFGF (10 ng/ml) wasalso added to the culture medium which already contained SCGF and VEGF.The tubes prepared in this way were then centrifuged for 6 hours at 12 gand 37° C. Subsequently, the coated CardioFix cylinders were carefullytransferred into a 25 cm² culture flask with 10 ml IMDM+10% FCS+10%horse serum+10⁻⁶ mol/l hydrocortisone+VEGF (50 ng/ml)+bFGF (10ng/ml)+IGF-1 (10 ng/ml) and cultivated for 8 weeks. The cylinder wasthen analysed immunohistochemically. A confluent monolayer of cells withan endothelial cell morphology, which were immunohistochemicallypositive for vWF and VE-cadherin, was detected.

Example 8

Diagnosis of Metastases by Means of Radioactively Labelled EndothelialProgenitor Cells

In this example, the AC133+cells were cultivated for 14 days at a celldensity of 2×10⁶ cells/ml in IMDM+10% FCS+10% horse serum+10⁻⁶ mol/lhydrocortisone+Flt3 ligand (50 ng/ml)+SCGF (100 ng/ml)+VEGF (50 ng/ml)and subsequently for a further 4 days under the influence of SCGF andVEGF. The cells were trypsinised, washed in PBS and again taken up inculture medium. In this case, the medium was supplemented with 20 U/mlof heparin and the cells were incubated for 15 minutes at roomtemperature. To the medium, 50 MBq of 18F-fluorodeoxyglucose were addedand the cells were incubated for a further 30 minutes at 37° C. Thelabelling reaction was then stopped by adding PBS. The cells werecentrifuged for 10 minutes at 450 g, washed twice in PBS and resuspendedin 100 μl/2×10⁸ cells of 0.9% NaCl solution. Subsequently, theradioactively labelled cells were injected into the tail vein of tumourcarrying naked rats. Each animal received 2×10⁸ labelled cells.Measuring of the animals took place after 30, 60, 90 and 120 minutes bypositron emission tomography. In this case, a maximum build-up of thelabelled cells in the tumour tissue was found after 90 minutes.

Abbreviations Used:

-   SCGF stem cell growth factor-   SCF stem cell factor-   VEGF Vascular endothelial growth factor; according to the invention,    the use of the isoforms A, B, C and/or D is included.-   EGF epidermal growth factor-   aFGF acidic fibroblast growth factor-   bFGF basic fibroblast growth factor-   IGF-1 insulin-like growth factor-1-   NGF nerve growth factor-   TGF-β1 transforming growth factor-β1-   Flt3 ligand fetal liver tyrosine kinase 3 ligand-   G-CSF granulocyte colony-stimulating factor-   GM-CSF granulocyte-macrophage colony-stimulating factor-   M-CSF macrophage colony-stimulating factor-   CTNF ciliary neurotrophic factor-   KGF keratinocyte growth factor-   IL-1, -3, -6, -11 Interleukin-1, -3, -6, -11-   PlGF placenta-like growth factor-   TNFα tumor necrosis factor α-   PD-ECGF platelet-derived endothelial-cell growth factor-   TPO thrombopoietin-   EPO erythropoietin-   AP-1, -2 angiopoietin-1, -2-   LIF leukemia inhibiting factor-   ECGS endothelial cell growth supplement-   CEACAM CEA-related cell adhesion molecule-   HGF hepatocyte growth factor-   GDNF glial cell line-derived neurotrophic factor-   BDNF brain-derived neurotrophic factor-   PDGF-BB platelet-derived growth factor-bb-   BMP-4 bone morphogenetic protein-4-   IMDM Iscove's modified Dulbecco's medium-   MEM minimum essential medium-   RPMI RPMI 164b medium-   EGM-2 endothelial growth medium-2-   GFP green fluorescent protein-   DMSO dimethyl sulfoxide-   HSA Human serum albumin-   FBS Fetal calf serum

REFERENCES

-   SCGF Hiraoka et al., Proc. Natl. Acad. Sci. USA 94, 7577-7582, 1997-   SCF Rottapel et al., Mol. Cell. Biol. 11, 3043-3051, 1991-   VEGF Houck et al., Mol. Endocrinol., 5, 1806-1814, 1991-   EGF Mc Clure et al., J. Cell. Physiol. 107, 195-207, 1981-   aFGF Wang et al., Mol. Cell. Biol. 9, 2387-2395, 1989-   bFGF Maciag et al., Proc. Natl. Acad. Sci. USA 76, 5674-5678, 1979-   Pleiotrophin Merenmies J, Rauvala H, J. Biol. Chem. 265, 1621-1624,    1990;    -   Tesuka et al., Biochem. Biophys. Res. Commun. 173, 246-251,        1990;    -   Li et al., Science 250, 1690-1694, 1990-   PlGF Maglione et al., Proc. Natl. Acad. Sci. USA 88, 9267-9271, 1991-   TGFα Lee et al., Nature 313, 489-491, 1985-   TNFα Shirai et al., Nature 313, 803-806, 1985;    -   Wang et al., Science 228, 149-154, 1985;    -   Marmenout et al., Eur J. Biochem. 152, 515-522, 1985-   Angiogenin Kurachi et al., Biochemistry 24, 5494-5499, 1985-   PD-ECGF Miyazono K, Heldin C H, Biochemistry 28, 1704-1710, 1989;    -   Hagiwara et al., Mol. Cell Biol. 11, 2125-2132, 1991-   Proliferin Parfett et al., Mol. Cell Biol. 5, 3289-3292, 1985;    -   Fassett et al., Endocrinology 141, 1863-1871, 2000-   IL-1 Mc Kernan et al., Cell Immunol. 80, 84-96, 1983-   IL-3 Yang et al., Cell 47, 3-10, 1986-   IL-6 Fiers et al., Immunol. Lett. 16, 219-226, 1987-   IL-11 Paul et al., Proc. Natl. Acad. Sci. USA 87, 7512-7519, 1990-   TPO Lok et al., Nature 369, 565-568, 1994    -   Bartley et al., Cell 77, 1117-1124, 1994    -   de Sauvage et al., Nature 369, 533-538, 1994    -   Sohma et al., FEBS Lett. 353, 57-61, 1994-   EPO Gasson et al., Nature 315, 768-771, 1985-   AP-1 Davis et al., Cell 87, 1161-1169, 1996-   AP-2 Kim et al., J. Biol. Chem. 275, 18550-1856, 2000-   LIF Gearing et al., EMBO J. 6, 3995-4002, 1987-   CEACAM Ergün et al., Mol. Cell 5, 311-320, 2000-   HGF Nakamura et al., Nature 342, 440-443, 1989-   GDNF Lin et al., J. Neurochem. 63, 758-768, 1994    -   Suranto et al., Hum. Mol. Genet. 6, 1267-1273, 1997-   BDNF Leibrock et al., Nature 341, 149-152, 1989-   PDGF-BB Cook et al., Biochem. J. 281, 57-65, 1992-   BMP-4 Kurihara et al., Biochem. Biophys. Res. Comm. 192, 1049-1056,    1993-   Neutrophin Hohn et al., Nature 344, 339-341, 1990-   CNTF Negro et al., Eur. J. Biochem. 201, 289-294, 1991-   Anti-CD7 mAb Foa et al., Cell Immunol. 89, 194-201, 1984-   Anti-CD31 mAb Newman P J et al., Science 247, 1219-1222, 1990-   Anti-CD34 mAb Sovalat et al., Hematol. Cell Ther. 40, 259-268, 1998-   Anti-CD45 mAb Hall L R et al., J. Immunol. 141, 2781-2787, 1988-   Anti-CD54 mAb Maio et al., Blood 76, 783-790, 1990-   Anti-CD90 mAb Palmer et al., Clin. Exp. immunol. 59, 529-538, 1985-   Anti-CD114 mAb Toba et al., Cytometry 35, 249-259, 1999-   Anti-CD116 mAb Toba et al., Cytometry 35, 249-259, 1999-   Anti-CD117 mAb Ricotti et al., Blood 91, 2397-2405, 1998-   Anti-CDw123 mAb Toba et al., Cytometry 35, 249-259, 1999-   Anti-CD127 mAb de Waele et al., Eur. J. Hematol. 66, 178-187, 2001-   Anti-CD133 mAb Miraglia et al., Blood 90, 5013-5021, 1997-   Anti-CD135 mAb Rappold et al., Blood 90, 111-125, 1997-   Anti-CD140b mAb Foss et al., Eur. J. Hematol. 66, 365-376, 2001-   Anti-CD144 mAb Joop et al., Thromb. Hemost. 85, 810-820, 2001-   Anti-CD164 mAb Zannatino et al., Blood 92, 2613-2828, 1998-   Anti-CD172a mAb Seiffert et al., Blood 94, 3633-3643, 1999-   Anti-CD173 mAb Cao et al., Glycobiology 11, 677-683, 2001-   Anti-CD174 mAb Cao et al., Glycobiology 11, 677-683, 2001-   Anti-CD175 mAb Doyle et al., Hum. Genet. 33, 131-146, 1976-   Anti-CD176 mAb Karsten et al., Hybridoma 14, 37-44, 1995-   Anti-CD184 mAb Tanaka et al., J. Virol. 75, 11534-11543, 2001-   Anti-CD201 mAb Tsuneyoshi et al., Thromb. Hemost. 85, 356-361, 2001-   Anti-CD202b mAb William et al., Circ. res. 87, 370-377, 2000-   Anti-CD224 mAb Petersen et al., Hepatology 27, 433-445, 1998-   Anti-CD227 mAb Brossart et al., Cancer Res. 61, 6846-6850, 2001-   Anti-CD228 mAb Johnson et al., Hybridoma 1, 381-385, 1982-   Anti-CD243 mAb Del Poeta et al., Leuk. Res. 23, 451-465, 1999-   Anti-EGF-R mAb Ramos-Suzarte et al., J. Nucl. Med. 40, 768-775, 1999-   Anti-FGF-R2 mAb Wu et al., Acta Obstet. Gynecol. Scand. 80, 497-504,    2001-   Anti-P1H12 mAb Solovey et al., N. Engl. J. Med. 337, 1584-1590, 1997-   Anti-KDR mAb Hunt et al., Curr. Opin. Mol. Ther. 3, 418-424, 2001    -   Michel et al., Hear Res. 155, 175-180. 2001-   Anti-EN4 mAb Burgio et al., Clin. Exp. Immunol. 96, 170-176, 1994-   Anti-BENE mAb de Marco et al., J. Biol. Chem. 276, 23009-23017, 2001-   Gentransfection by Wahlers et al., Gene Ther. 8, 477-486, 2001 means    of vectors-   Genetransfection Gubin et al., Biotechniques 27, 1162-1164, 1999 by    means of liposomes-   Gene transfection Cui et al., Gene Ther. 8, 1508-1513, 2001 by means    of naked DANN-   Gene transfection Ear et al., J. Immunol. Methods 257, 41-49, 2001    by means of electroporation

1. Method for carrying out in vitro expansion of multipotent stem cellscharacterised in that multipotent stem cells are cultivated in thepresence of Flt3 ligand and at least one growth factor from the groupconsisting of SCF, SCGF, VEGF, bFGF, insulin, NGF and TGF-β.
 2. Methodaccording to claim 1 characterised in that the following growth factorsare used: a) Flt3 ligand and VEGF, b) Flt3 ligand, SCGF and VEGF, c)Flt3 ligand and EGF, d) Flt3 ligand, EGF and bFGF.
 3. Method accordingto claim 1 or 2 characterised in that IGF-1 and/or EGF is/areadditionally used.
 4. Method according to claims 1 to 3 characterised inthat multipotent stem cells are gene transfected during the expansion.5. Method for the in vitro expansion and differentiation of multipotentstem cells characterised in that a) in a first phase, a method accordingto claims 1 to 4 is carried out for the expansion of multipotent stemcells and b) the expanded cells are differentiated in a second phase,the cells being (i) cultivated for hematopoeitic differentiation in thepresence of G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-11, TPO and/or EPO,(ii) cultivated for endothelial differentiation in the presence of VEGF,aFGF, bFGF, ECGS, AP-1, AP-2, NGF, CEACAM, pleiotrophin, angiogenin,PlGF, and/or HGF, (iii) cultivated for mesenchymal differentiation inthe presence of PDGF-BB, TGF-β and/or BMP-4, (iv) cultivated forneuronal differentiation in the presence of NGF, CNTF, GDNF and/or BDNFor (v) cultivated for hepatocytic differentiation in the presence ofHGF.
 6. Process according to claim 5 characterised in that (i) IL-1, SCFand/or SCGF is/are also added for hematopoietic differentiation, (ii)LIF, EGF, IGF-1, PDGF, PDECGF, TGFα, TGFβ, TNFα, estrogen, proliferin,IL-3, G-CSF, GM-CSF, EPO SCF and/or SCGF is/are also added forendothelial differentiation, (iii) EGF, aFGF, bFGF, IGF-1, SCF and/orSCGF is/are also added for mesenchymal differentiation, (iv) EGF, bFGF,GF-1, IL-1b, Il-6, Il-11, LIF, Flt3 ligand, SCF and/or BMP-4 is/are alsoadded for neuronal differentiation or (v) EGF, IGF-1, insulin, HCG, KGF,TNF-α, Flt3 ligand, SCF and/or SCGF is/are also added for hepatocyticdifferentiation.
 7. Process according to claim 6 characterised in that,in the second phase (i) a combination of SCF, IL-3, IL-6, G-SCF, GM-CSFand EPO is used for hematopoietic differentiation, (ii) a combination ofSCGF and VEGF is used for endothelial differentiation, (iii) acombination of EGF, PDGF-BB, IGF-1, bFGF and BMP-4 is used formesenchymal differentiation, (iv) a combination of BDNF, GDNF, EGF andbFGF is used for neuronal differentiation or (v) a combination of Flt3ligand, SCF, HGF and TGF-β is used for hepatocytic differentiation. 8.Method for producing a pharmaceutical preparation characterised in thata method according to claims 1 to 4 is carried out and the multipotentstem cells obtained are processed for administration.
 9. Method forpreparing a pharmaceutical preparation characterised in that a methodaccording to claims 5 to 7 is carried out, progenitor cells and/ormatured cells being isolated and the cells obtained being processed foradministration.
 10. Method for preparing a pharmaceutical preparationcharacterised in that a) a method according to claims 1 to 4 is carriedout and b) a method according to claims 5 to 7 is carried out in whicheither progenitor cells and/or matured cells are isolated, the cellsobtained in a) and b) being mixed and processed for administration. 11.Method according to claims 8 to 10 characterised in that the cells areradioactively labelled.
 12. Method according to claims 8 to 11characterised in that the cells are taken up in 0.9% saline solution.13. Method for coating implantable materials in which a method accordingto claims 5 to 7 is carried out with endothelial differentiation of thecells, the material to be implanted being introduced during and/or atthe end of the differentiation phase into the cell-containing culturemedium in which the differentiation of the cells takes place.
 14. Methodaccording to claim 13 characterised in that the implantable materialsare coated with fibronectin.
 15. Method according to claim 13 or 14characterised in that the implantable materials are coronary stents orvascular valves.
 16. Use of the cells obtained according to the methodof claims 1 to 7 for producing artificial tissues.
 17. Use according toclaim 16 characterised in that the tissue is brain, liver, kidney,heart, cartilage, bone, retinal, muscle or connective tissue or skin.18. Method for producing artificial tissue characterised in that amethod according to claims 5 to 7 is carried out and that the expandedcells are brought into contact, before or during the differentiation,with a matrix to which the cells can attach.
 19. Process according toclaim 18 characterised in that the method is carried out withendothelial or mesenchymal differentiation and the matrix is broughtinto contact, during the differentiation phase, with endothelial ormesenchymal progenitor cells.
 20. Process according to claim 18 or 19characterised in that the tissue is brain, liver, kidney, heart,cartilage, bone, retinal, muscle or connective tissue or skin. 21.Process for producing artificial blood vessels characterised in that amethod according to claim 18 is carried out with endothelialdifferentiation and the cells are brought into contact with acylindrical matrix.
 22. Pharmaceutical composition obtained by a methodaccording to claims 8 to
 12. 23. Implantable material obtained by amethod according to claim 13 or
 14. 24. Implantable material accordingto claim 23 characterised in that it is a coronary stent or a vascularvalve.
 25. Artificial tissue which is produced by using the cellsobtained by a method of claims 1 to
 7. 26. Artificial tissue which isobtained by a method according to claim 18 or
 19. 27. Artificial tissueaccording to claim 25 or 26 characterised in that it is brain, liver,kidney, heart, cartilage, retinal, muscle or connective tissue or skin.28. Artificial blood vessel obtained by a method according to claim 21.